Abstract Uterine leiomyomas, also known as fibroids, are common benign smooth muscle tumors in women of reproductive age. Leiomyomas can cause symptoms that can significantly reduce the quality of life. Treatment mainly involves hysterectomy or myomectomy, the latter for women who wish to preserve their uterus. Most leiomyomas harbor one of the three driver mutations affecting either MED12, HMGA2, or FH. Recently identified rare subtypes include tumors with mutations in genes linked to the neddylation of the Cullin 3-RING E3 ligase or SRCAP complex. Recurrence of leiomyomas is poorly understood in relation to their clonal origins and molecular factors. The aim of this study was to characterize the mutational profiles of leiomyomas from recurrent operations and to identify the frequency of clonally related tumors. We utilized a retrospective cohort of 234 women who underwent laparoscopic or open abdominal myomectomy, with 46 (20%) of them experiencing multiple procedures related to leiomyomas. We examined the mutation profiles of 133 leiomyomas (62 index and 71 recurrent tumors) from 33 patients who required multiple tumor removal surgeries. We screened the tumors for the three primary leiomyoma drivers—MED12 mutations, HMGA2 overexpression, and FH-deficiency—to identify potentially clonal tumors. We found that 21 out of 33 (64%) patients had tumors from multiple operations with identical leiomyoma driver alterations. To further assess the clonal relationship, we executed whole exome sequencing on these 52 tumors. We identified three patients with two clonally related tumors each through shared somatic copy number alterations and point mutations. The clonally related tumors included HMGA2, FH, and wild-type tumors. Notably, leiomyomas with MED12 mutations—the most common molecular leiomyoma subtype—were not found among the clonally related tumors. Three patients harbored numerous FH-deficient tumors from recurrent operations. In all those patients, we found FH germline mutations, characteristic of Hereditary Leiomyomatosis and Renal Cell Cancer syndrome (HLRCC). Moreover, we identified somatic mutations in YEATS4, a member of the SRCAP complex, in four recurrent tumors from three patients. Interestingly, all YEATS4 mutations were different, and the tumors were not clonally related. Further research is required to elucidate the role of YEATS4 in leiomyoma recurrence. In conclusion, our systematic study confirms that reinterventions are common after myomectomy, and while uterine leiomyomas typically develop independently, some share a clonal origin. Additionally, we show that recurrence may be due to genetic predispositions, such as germline FH mutations. Citation Format: Sara Khamaiseh, Anna Äyräväinen, Maare Arffman, Siiri Reinikka, Annukka Pasanen, Ralf Bützow, Päivi Härkki, Pia Vahteristo. Genetic background of recurrent uterine leiomyomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5055.